Producing immunogenic constructs using soluble carbohydrates activated via organic cyanylating reagents

ABSTRACT

The invention relates to a process for producing an immunogenic construct comprising activating at least one first carbohydrate-containing moiety with CDAP, and covalently joining the activated first moiety to a second moiety. Preferably, the first moiety is a polysaccharide and the second moiety is a protein. Immunogenic constructs are prepared by this process using either direct or indirect conjugation of the first and second moieties.

GOVERNMENT INTEREST

The invention may be manufactured, licensed, and used for U.S.governmental purposes without the payment of any royalties to the patentowner thereon.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of U.S. Ser. No. 08/124,491, filed Sep.22, 1993, abandoned.

BACKGROUND OF THE INVENTION

Certain agents such as tetanus toxoid can innately trigger the immuneresponse, and may be administered in vaccines without modification.Other important agents are not immunogenic, however, and must beconverted into immunogenic molecules or constructs before they caninduce the immune response.

This invention relates generally to advantageous processes for makingimmunogenic constructs. The invention also relates to the resultingimmunogenic constructs and vaccines prepared therefrom, and the use ofsuch immunogenic constructs.

More specifically, the invention relates to methods of activatingcarbohydrate-containing antigens for use in preparing immunogenicconstructs. Immunogenic constructs are very advantageously prepared byactivating a carbohydrate-containing moiety with an organic cyanylatingagent such as 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate(CDAP).

A variety of cyanylating reagents are known per se, e.g., as reagentsfor activating insoluble particles to prepare gels for affinitychromatography. See Wilcheck et al., Affinity Chromatography. Meth.Enzymol., 104C:3-55. Wakelsman et al., J.C.S. Chem. Comm., 1976:21(1976), reported that CDAP is a mild reagent that can be used formodifying protein cysteine groups. Kohn et al., Anal. Biochem, 115:375(1981), compared CDAP, N-cyanotriethyl-ammonium tetrafluoroborate(CTEA), and p-nitrophenylcyanate (pNPC) as activating agents foragarose, an insoluble polysaccharide resin. Other researchers have usedCDAP to activate other types of insoluble particles, such as Sepharoseand glyceryl-controlled pore glass. See, e.g., Carpenter et al., Journalof Chromatography, 573:132-135 (1992).

U.S. Pat. No. 3,788,948 to Kagedal et al. generally describes a methodthat uses organic cyanate compounds to bind organic compounds containinga primary or secondary amino group to polymers containing one or morehydroxyl and/or primary and/or secondary amino groups, e.g., to bindwater-soluble enzymes to water-insoluble polymers. Kagedal et al.describe a method using certain organic cyanate compounds such as pNPChaving advantages over cyanogen bromide.

Similarly, Andersson et al., International Journal of Cancer, 47:439-444(1991), report using CDAP to activate a soluble polysaccharide prior toconjugation with protein. They directly conjugated epidermal growthfactor (EGF) to low molecular weight 40 kDa dextran activated withcyanate, and used very high dextran to EGF ratios of approximately 50:1(wt./wt.) to produce dextran-EGF conjugates and studied the binding ofthis conjugate to cultured cells.

Kagedal et al. and Andersson et al., however, are not concerned withimmunogenic constructs. Indeed, conjugates of proteins to low molecularweight dextrans have been reported to be poorly or non-immunogenic. T.E. Wileman, J. Pharm. Pharmacology, 38:264 (1985).

The degree of immunogenicity, of course, is an important property ofimmunogenic constructs for vaccination purposes. The process ofvaccination employs the body's innate ability to protect itself againstinvading agents by immunizing the body with antigens that will not causethe disease but will stimulate the formation of antibodies, cells, andother factors that will protect against the disease. For example, deadorganisms are injected to protect against bacterial diseases such astyphoid fever and whooping cough, toxoids are injected to protectagainst tetanus and diphtheria, and attenuated organisms are injected toprotect against viral diseases such as poliomyelitis and measles.

It is not always possible, however, to stimulate antibody formationmerely by injecting the foreign agent. The vaccine preparation must beimmunogenic, that is, it must be able to induce an immune response. Theimmune response is a complex series of reactions that can generally bedescribed as follows: (i) the antigen enters the body and encountersantigen-presenting cells that process the antigen and retain fragmentsof the antigen on their surfaces; (ii) the antigen fragments retained onthe antigen-presenting cells are recognized by T cells that provide helpto B cells; and (iii) the B cells are stimulated to proliferate anddivide into antibody-forming cells that secrete antibodies against theantigen.

Antibodies to most bacterial polysaccharides have been shown to provideprotection against infection with encapsulated bacteria. The inabilityof newborns and infants to mount vigorous responses to T-cellindependent (TI) antigens, as exemplified by polysaccharides, hasresulted in their extreme susceptibility to life-threatening infectionswith these organisms. This impaired immune response to TI antigens canbe overcome by conjugating T-cell epitopes onto the polysaccharides,thereby converting them into T-cell dependent (TD) antigens.

There are two conjugation methods generally used for producingimmunogenic polysaccharide constructs: (1) direct conjugation ofcarbohydrate and protein; and (2) indirect conjugation of carbohydratesand protein via a bifunctional linker or spacer reagent. Generally, bothdirect and indirect conjugation require chemical activation of thecarbohydrate moiety prior to its derivatization.

Chemical activation refers to the conversion of a functional group to aform that can undergo additional chemical reactions, e.g., the additionof a functional group or of a large moiety such as a protein.Derivatization is the addition of functional chemical group(s) or spacerreagent(s) to a protein.

Unfortunately, artisans have encountered a number of problems in formingimmunogenic constructs via conjugation using activation methods. Forexample, the production of conjugate vaccines has been a formidablechallenge, in part, because of the difficulty in activating thepolysaccharide and conjugating the protein under conditions that do notlead to their degradation or to the destruction of their immunogenicepitopes. In preparing immunogenic constructs, the method used should besufficiently gentle to retain important antigenic sites, i.e., epitopes,on the molecules. Thus, it is desirable to maintain the integrity of thestructure and to preserve epitopes in these compounds. Unfortunately,the preparation steps currently used in the art are frequently notgentle and can destroy native carbohydrate and/or protein structures.

Moreover, many of the known techniques for carbohydrate modificationrequire anhydrous conditions. Unfortunately, however, carbohydrates arefrequently insoluble in organic solvents. Marburg et al., J. Amer. Chem.Soc., 108:5282 (1986).

Thus, although there is a large body of chemical literature describingthe modification of carbohydrates, much of it is unsuitable for use withaqueous-based antigens. One approach has been the modification ofpolysaccharides to enhance their solubility in organic solvents. Forexample, by replacing the acidic hydrogen on certain acidicpolysaccharides with the hydrophobic tetrabutyl ammonium counter-ion,Marburg et al. were able to solubilize polysaccharides in organicsolvents and activate hydroxyls with carbonyl diimidazole, a reagentwhich must be used in dry solvent. This method is used withpolysaccharides, such as Haemophilus influenzae PRP and Pneumococcalpolysaccharides type 6B and 19F. Coupling of proteins can also beachieved through reductive amination, either using the aldehyde found onthe reducing end of the polysaccharide or created by oxidation of thecarbohydrate. Both of these approaches have intrinsic limitations and,thus, for high molecular weight polysaccharides, coupling through thereducing end is usually slow and inefficient and oxidation often resultsin cleavage of the polysaccharide chain or otherwise affects theantigen.

Certain carbohydrates contain groups, such as amino or carboxyl groups,that can be more easily activated or derivatized before conjugation. Forinstance, the amino groups in Pseudomonas Fisher Type I can be easilyderivatized with iodoacetyl groups and bound to a thiolated protein. Thecarboxyl groups in carbohydrates such as Pneumonococcal type III can beeasily activated with water-soluble carbodiimides, such as EDC, and canthen be coupled directly to protein. Unfortunately, however, this groupof carbohydrates is limited.

Other carbohydrates have aldehyde groups at the terminal reducing endthat can be exploited for derivatization and conjugation. It is alsopossible to create aldehyde groups with oxidizing reagents, e.g., sodiumperiodate. Aldehyde groups can be condensed with amino groups on proteinor with a bifunctional linker reagent. This condensation reaction,especially with the terminal reducing end of a high molecular weightpolysaccharide, however, often proceeds quite slowly and inefficiently.This is exacerbated when directly conjugating carbohydrate aldehydes toproteins. Thus, yields are often very low using this method. Moreover,sodium periodate may break up carbohydrates into smaller fragmentsand/or disrupt epitopes, which may be undesirable.

Most carbohydrates must be activated before conjugation, and cyanogenbromide is frequently the activating agent of choice. See, e.g., Chu etal., Inf. & Imm., 40:245 (1983), and Dick & Beurret, "Glycoconjugates ofBacterial Carbohydrate Antigens," Conjugate Vaccines, J. M. Cruse & R.E. Lewis (eds.), vol. 10, 48-114 (1989). The first licensed conjugatevaccine was prepared with CNBr to activate HIB PRP, which was thenderivatized with adipic dihydrazide and coupled to tetanus toxoid usinga water-soluble carbodiimide.

To briefly summarize the CNBr-activation method, cyanogen bromide isreacted with the carbohydrate at a high pH, typically a pH of 10 to 12.At this high pH, cyanate esters are formed with the hydroxyl groups ofthe carbohydrate. These, in turn, are reacted with a bifunctionalreagent, commonly a diamine or a dihydrazide. These derivatizedcarbohydrates may then be conjugated via the bifunctional group. Incertain limited cases, the cyanate esters may also be directly reactedto protein.

The high pH is necessary to ionize the hydroxyl group because thereaction requires the nucleophilic attack of the hydroxyl ion on thecyanate ion (CN⁻). As a result, cyanogen bromide produces many sidereactions, some of which add neo-antigens to the polysaccharides. M.Wilcheck et al., Affinity Chromatography. Meth. Enzymol., 104C:3-55.More importantly, many carbohydrates or moieties such as HIB PRP and Pn6can be hydrolyzed or damaged by the high pH necessary to perform thecyanogen bromide activation.

Another problem with the CNBr-activation method is that the cyanateester formed is unstable at high pH and rapidly hydrolyzes, reducing theyield of derivatized carbohydrate and, hence, the overall yield ofcarbohydrate conjugated to protein. Many other nonproductive sidereactions, such as those producing carbamates and linearimidocarbonates, are promoted by the high pH. Kohn et al., Anal.Biochem, 115:375 (1981). Moreover, cyanogen bromide itself is highlyunstable and spontaneously hydrolyzes at high pH, further reducing theoverall yield.

Furthermore, the cyanogen bromide activation is difficult to perform andunreliable. Cyanogen bromide is highly toxic and potentially explosive.Extreme care must be used when working with large quantities as used inmanufacture. All operations must be carried out in a suitable fumehood.It is also known to those in the art that the activation is not easilyreproducible because some batches of cyanogen bromide work well and somedo not. Cyanogen bromide is also poorly soluble in water, making itdifficult to control the amount of soluble cyanogen bromide available toreact with the carbohydrate. Even use of the same batch of cyanogenbromide and apparently identical reaction conditions do not always leadto identical results.

In addition to these disadvantages, it is very difficult to control thedegree of carbohydrate activation achieved by using cyanogen bromide. Itis also very difficult to achieve a high level of carbohydrateactivation using this method. Increasing the amount of cyanogen bromidepresent is ineffective and only leads to increased side reactionswithout an increase in activation. Kohn et al., Applied Biochem andBiotech, 9:285 (1984).

Thus, while cyanogen bromide activation has proven to be a very usefulreagent, it has a number of limitations. For example, cyanogen bromiderequires a high pH (10-12) in order to make the hydroxyls sufficientlynucleophilic to react with the cyanate ion. However, neither CNBr northe cyanate ester intermediate is stable at high pH, and consequentlymost of the reagent either hydrolyzes or undergoes nonproductive orunwanted side reactions. Thus, the efficiency of polysaccharideactivation is low. Furthermore, the high pH required for activation canhydrolyze or damage many pH-sensitive polysaccharides. In addition, CNBris toxic and difficult to work with in small quantities.

Moreover, as noted above, other conjugation methods suffer from variousdrawbacks. For example, although polysaccharides such as Cryptococcusneoformans and Pneumococcal polysaccharide type 3 and VI antigen havecarboxyl groups that can be activated with carbodiimides in preparationfor coupling to a protein, and polysaccharides such as PseudomonasFisher type III have amino groups that can be conveniently used, theseantigens form a relatively limited group of all polysaccharides. Otherapproaches are therefore needed to activate or functionalize themajority of polysaccharides.

Thus, there is a need in the art for a method to produce immunogenicconstructs that is gentle, maintains the integrity of the structure ofthe carbohydrates and proteins, preserves epitopes in the compounds, iseasy to perform, is reliable, is readily reproducible, is readily scaledup, and works with a wide variety of polysaccharides.

SUMMARY OF THE INVENTION

An object of the invention is to achieve a gentle method for producingimmunogenic constructs. Another object is to arrive at a method formaking immunogenic constructs that maintains the integrity of thestructure of the carbohydrates and proteins, and preserves epitopes inthe compounds. An additional object is to achieve a method ofmanufacturing immunogenic constructs that is easy to perform, reliable,and readily reproducible. A further object is to develop a method formaking immunogenic constructs that may be used with a variety ofpolysaccharides. An additional object is to obtain a convenient methodfor making soluble conjugate vaccines. Another object is to attain amethod that is easily scaled up. These and other objects and advantagesof the invention will be apparent from the detailed description below.

The present invention attains the above objects, thereby overcoming theproblems and disadvantages of known methods for producing immunogenicconstructs, by a conjugation process that employs a carbohydrateactivation method that is safe, easy, inexpensive, and gentle tocarbohydrates. Moreover, the method advantageously employs a homogeneousreaction.

The method of the present invention advantageously uses an organiccyanylating reagent, most preferably1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (CDAP), toactivate carbohydrate-containing moieties. Using the inventive method, aconjugate of a polysaccharide and protein can be prepared where only thepolysaccharide is modified, making it possible to recover the protein.Moreover, a conjugate of water-soluble and/or surfactant-solublemoieties may be readily prepared according to the invention.

In one preferred embodiment, the invention comprises directlyconjugating the activated carbohydrate-containing moiety to a secondmoiety, such as a water-soluble protein. In another preferredembodiment, the method of the invention comprises covalently binding afunctional (bifunctional or heterofunctional) reagent to the activatedcarbohydrate-containing moiety, and further reacting the functionalreagent with the second moiety, e.g., a T-dependent antigen, to form aconjugate immunogenic construct, wherein the carbohydrate-containing andTD moieties are linked by the spacer or linker formed by the functionalreagent.

In another preferred embodiment, the immunogenic construct is adual-carrier construct of a type described in related U.S. patentapplication Ser. No. 07/834,067, filed Feb. 11, 1992 (now abandoned),and its continuation-in-part, Ser. No. 08/055,163, filed Feb. 10, 1993(now abandoned), the specifications of which are incorporated byreference herein. Exemplary primary carriers for such a constructinclude Pneumococcal type 14 (Pn14) and DNA polymers.

The invention is advantageously applicable to a wide variety of solublecarbohydrate-containing moieties, which after activation with CDAP maybe either directly conjugated to protein or indirectly conjugated toprotein through a spacer or a linker. The invention enables others toproduce more effective immunogenic constructs more efficiently and lessexpensively than immunogenic constructs prepared using known methods.

Moreover, because CDAP and reaction conditions are so gentle, the riskof destruction of carbohydrate structure and, hence, destruction ofnaturally-occurring epitopes, is greatly diminished. Furthermore, themethod has the advantages summarized in Table 1 below in comparison withthe presently used method employing cyanogen bromide.

                  TABLE 1    ______________________________________    Comparison of Carbohydrate Activation    in the Synthesis of Conjugates    CNBr              CDAP    ______________________________________    High pH (10-12)   Near neutral or mildly basic                      pH (e.g., 7-9)    Destroys many CHO epitopes                      Little or no alteration of                      CHO epitopes    High toxicity (fume-                      Low toxicity    hood required)    Dangerous in large                      Safe in large quantities    quantities    Difficult to work with                      Easy to work with small    small quantities  quantities    Low yields        High yields    Multiple side reactions                      Minimal or no side reactions    Does not easily permit                      Allows direct conjugation to    direct conjugation to                      protein and enables recovery    protein           of unconjugated protein    Batch-to-batch variation                      Reproducible    ______________________________________

Additional advantages to using CDAP are that it can be prepared inadvance and stored in a solution for several months, and theconcentration of active reagent can be easily determined from itsabsorbance at 301 nm (Kohn et al., Anal. Biochem, 115:375 (1981)). Thismakes it possible to standardize the reagent concentration and makes thecarbohydrate derivatization more reproducible, which is important forits use in vaccine preparation.

The above-mentioned advantages apply both to the direct conjugation ofproteins to carbohydrates and to indirect conjugation via a spacer.Additional objects and advantages of the invention will be apparent fromthe detailed description and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an example of a generalized scheme for the activation ofcarbohydrate using organic cyanylating reagents.

FIG. 2 depicts an exemplary scheme for conjugation of an activatedcarbohydrate to protein, with direct conjugation shown at the bottomleft-hand side and indirect conjugation using a bifunctional reagentshown at the bottom right-hand side.

FIG. 3 shows a model of an immunogenic construct.

FIG. 4 illustrates the incorporation of NH₂ groups into dextran versusthe moles of CDAP added per mole of dextran at 10 mg/ml dextran.

FIG. 5 illustrates the elution profile of a ³ H-BSA-dextran conjugatefrom a S400SF gel filtration column.

FIG. 6 illustrates the OD280 absorbance of immunogenic constructsprepared according to the method of the invention, eluted from S400SFgel filtration column.

FIG. 7 illustrates the elution profile of Hδ^(a) /1-(CDAP)-dextran fromS400SF gel filtration column.

FIG. 8 illustrates OD280 and OD430 values of column samples eluted fromS400SF gel filtration column loaded with Hδ^(a) /NH₂ -(CDAP)-dextran.

FIG. 9 illustrates the immunoreactivity of immunogenic constructsprepared using the methods of the invention.

FIG. 10 shows the results of derivatization of dextran (dex) with hexanediamine with CDAP (NH₂ /100 kDa dex versus mg CDAP/mg dex) at 1.6 mg/mldextran.

FIG. 11 is a graph of the efficiency of CDAP activation versus thepolysaccharide concentration.

FIG. 12 shows the direct conjugation of BSA to dextran for variousCDAP:polysaccharide ratios for CDAP activation.

FIG. 13 is a plot of the BSA/dextran ratio versus the time of additionof protein to CDAP-activated dextran.

FIG. 14 shows the stability of CDAP in water.

FIG. 15 illustrates the kinetics of protein coupling to CDAP-activatedpolysaccharide.

FIG. 16 shows the effect of pH on CDAP activation.

FIG. 17 is a bar graph showing the effect of pH and various buffers onthe coupling of BSA to CDAP-activated dextran.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

A generalized scheme for the activation of carbohydrates using organiccyanylating reagents (which may be represented generally by the formulaR--CN or {R⁺ --CN}X⁻, where R is an organic moiety and X is acounter-ion) is shown in FIG. 1. FIG. 2 illustrates the conjugation ofan activated carbohydrate to protein.

As used herein, "immunogenic construct" refers to an entity that canstimulate the immune response. The immunogenic construct comprises atleast one first moiety conjugated to at least one second moiety. As usedherein, a "moiety" is any substance that can be used to stimulate theimmune system either by itself or upon being coupled.

Exemplary moieties include carbohydrates, synthetic polymers such aspolyvinyl alcohol, proteins and glycoproteins, peptides, other antigens,adjuvant molecules, haptens, DNA, and combinations and derivativesthereof. Haptens refer to small molecules, such as chemicals, dust, andallergens, that by themselves are not able to elicit an antibodyresponse, but can once they are coupled to a carrier, e.g., TNP. Anantigen is any molecule that, under the right circumstances, can inducethe formation of antibodies. These haptens and antigens may derive frombut are not limited to bacteria, rickettsiae, fungi, viruses, parasites,drugs, or chemicals. They may include, for example, small molecules suchas peptides, oligosaccharides (e.g., the polyribosyl-ribitol-phosphateoligomers of H. influenzae), DNA oligomers, lipids, toxoids, endotoxin,etc. Preferred moieties are soluble in water or solubilized insurfactant.

In a preferred embodiment, the first moiety is a carbohydrate-containingmoiety. As used herein, "carbohydrate" means any soluble monosaccharide,disaccharide, oligosaccharide, or polysaccharide. Preferably, the firstmoiety is a polysaccharide, more preferably a water-solublepolysaccharide. Preferred polysaccharides include those listed in thechart below of exemplary vaccines.

The carbohydrate-containing moiety is preferably naturally occurring, asemisynthetic, or a totally synthetic large molecular weight molecule.In a preferred embodiment, at least one carbohydrate-containing moietyis selected from E. coli polysaccharides, S. aureus polysaccharides,dextran, carboxymethyl cellulose, agarose, Pneumococcal polysaccharides(Pn), Ficoll, Cryptococcus neoformans, Haemophilus influenzae PRP, P.aeroginosa, S. pneumoniae, lipopolysaccharides, Group A and Bstreptococcus, N. meningitidis, and combinations thereof.

In an especially preferred embodiment, the carbohydrate-containingmoiety is a dextran. As used herein, "dextran" (dex) refers to apolysaccharide composed of a single sugar, which may be obtained fromany number of sources (e.g., Pharmacia). Another preferredcarbohydrate-containing moiety is Ficoll, which is an inert,semisynthetic, non-ionized, high molecular weight polymer.

The carbohydrate-containing moiety is activated using an organiccyanylating reagent. Preferred organic cyanylating reagents are1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (CDAP),N-cyanotriethylammonium tetrafluoroborate (CTEA), andp-nitrophenylcyanate (pNPC). Of these reagents, CDAP is the mostpreferred. Other organic complexes with the cyanate group, optionallywith a variety of counter-ions, may be used. Particularly preferredorganic cyanylating reagents are those with non-nucleophiliccounter-ions such as tetrafluoroborate.

After activation via the organic cyanylating reagent, the first moietyis conjugated to the second moiety. Preferably, the second moiety is aprotein, which may be selected from viral, bacterial, parasitic, animal,and fungal proteins. Especially preferred second moieties includelipoproteins, bovine serum albumin (BSA), tetanus toxoid (TT), pertussistoxoid (PT), diphtheria toxoid (DT), heat shock protein, T-cellsuperantigens, and bacterial outer-membrane protein, all of which may beobtained from biochemical or pharmaceutical supply companies or preparedby standard methodologies (see, e.g., J. M. Cruse & R. E. Lewis, (eds.),Conjugate Vaccines in Contributions to Microbiology and Immunology, vol.10 (1989), which is incorporated herein by reference). Other suitableproteins may be selected from those known in the art.

Other preferred embodiments of the second moiety are albumin, a toxoid,a peptide, a T-cell or B-cell adjuvant, or any other compound capable ofactivating and recruiting T-cell help. The second moiety may be aT-dependent antigen as represented in FIG. 3.

The second moieties of the invention are capable of being conjugated toat least one carbohydrate-containing moiety. The second moieties mayeither contain functional groups that can react with thecarbohydrate-containing moiety or can be chemically manipulated to becapable of reacting with the carbohydrate-containing moiety.

Numerous copies of specific second moieties as well as a variety ofsecond moieties may be conjugated to the carbohydrate-containing moiety.Coupling of multiple copies of the second moiety to the first moietysignificantly augments antibody production to the second moiety.

The inventive process allows one to advantageously control the physicaland chemical properties of the immunogenic construct. In accordance withthe invention, the artisan may advantageously: modify the charge on thefirst and second moieties (an advantage in light of evidence thatcationized proteins may be more immunogenic); control the size of theconstruct by varying the size of the carbohydrate-containing moiety;select the degree of crosslinking of the inter- and intra-chainconstruct (to obtain variations of size and of the three-dimensionalmatrix); control the number of copies of the second moiety conjugated tocarbohydrate-containing moieties; and target to selected cellpopulations (such as to macrophages to enhance antigen presentation).Dick & Beurret, "Glycoconjugates of Bacterial Carbohydrate Antigens,"Conjugate Vaccines, J. M. Cruse & R. E. Lewis (eds.), vol. 10, 48-114(1989).

The immune response to the construct of the invention may be furtherenhanced by the addition of immunomodulators and/or cell-targetingmoieties. These entities include, for example, (1) detoxifiedlipopolysaccharides or derivatives, (2) muramyl dipeptides, (3)carbohydrates, lipids, and peptides that may interact with cell surfacedeterminants to target the construct to immunologically relevant cells,(4) interleukins, (5) antibodies, and (6) DNA oligomers.

Thus, in alternative embodiments, third moieties may be conjugated toone or more of the first and/or second moieties using methods such asCDAP activation as described herein or other known techniques. U.S.patent application Ser. Nos. 07/834,067 and 08/055,163 (bothapplications now abandoned) describe conjugation that promotes enhancedantibody responses to the third moiety. Certain techniques to conjugatevarious moieties to either the first or second moieties are well knownto those skilled in the art, e.g., involving coupling through availablefunctional groups (such as amino, carboxyl, thio and aldehyde groups).See S. S. Wong, Chemistry of Protein Conjugate and Crosslinking CRCPress (1991), and Brenkeley et al., "Brief Survey of Methods forPreparing Protein Conjugates With Dyes, Haptens and Cross-LinkingAgents," Bioconjugate Chemistry, 3:1 (Jan. 1992), which are incorporatedherein by reference. Thus, monofunctional reagents may be used as thirdmoieties, e.g., to modify the charge, change the hydrophobicity, labelthe construct, etc.

In the method of the invention, the carbohydrate-containing moiety isactivated using an organic cyanylating reagent. The organic cyanylatingreagent is preferably CDAP, which increases the electrophilicity of thecyanate and, when reacted with carbohydrate-containing moieties,transfers a cyano group to the hydroxyl groups of the carbohydrate, thuspreparing it for further reaction, i.e., direct or indirect conjugationto protein. The activation reaction can be carried out at neutral pH orunder mildly basic conditions (e.g., a pH of about 8 to about 10), whichimproves the stability and integrity of the polysaccharide and theactive intermediate.

CDAP is advantageous because it is highly stable and is relatively safe.CDAP is a water-soluble organic cyanylating reagent in which theelectrophilicity of the cyano group is increased, advantageouslypermitting the cyanylation reaction to be performed under mildconditions. Furthermore, CDAP can be used to activate a wide variety ofpolysaccharides, which can then be functionalized with diamines ordihydrazides. The high levels of activation and mild conditions of theCDAP cyanylation reaction permit proteins to be directly conjugated topolysaccharides in a one-pot reaction, thereby simplifying thepreparation of conjugate vaccines that induce antibody responses to boththe polysaccharide and the protein components, even in the absence of aspacer molecule. The ease of use of CDAP facilitates the preparation ofprotein-polysaccharide conjugate vaccines under a variety of conditions,thus making possible the study of the important parameters of theimmunogenicity of conjugate vaccines. Moreover, CDAP-activatedpolysaccharides can be used to prepare a variety of other usefulimmunological reagents, e.g., biotinylated polysaccharides andantibody-linked dextrans such as Hδ^(a) /1.

The activation is preferably performed at a pH of from about 6 to about10, more preferably of from about 9 to about 10. The pH may be adjustedby a variety of techniques (e.g., using a buffer, adding NaOH, etc.) tosuit the particular construct being prepared. For example, theactivation may be carried out in a variety of solvents using one or moreof a variety of suitable non-nucleophilic buffers known in the art.Suitable solvents include saline, water, and some organic solvents.Examples of suitable non-nucleophilic buffers include triethyl amine(TEA), 4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid (HEPES),phosphate, carbonate, and borate. Preferably, triethyl amine (TEA) isused as a buffer.

In a preferred embodiment of the invention, CDAP is dissolved in a stocksolution at a concentration of 100 mg/ml in dry acetonitrile or up to 75mg/ml in water. Depending on the nature of the carbohydrate-containingmoiety used and the degree of activation desired, various amounts ofCDAP may be optimal.

In a preferred embodiment, the concentration of thecarbohydrate-containing moiety is from 1 to 20 mg/ml, more preferablyfrom 1 to 15 mg/ml. The activation reaction can be performedsuccessfully with concentrations of carbohydrate-containing moiety up toabout 100 mg/ml.

Preferably, the CDAP to carbohydrate-containing moiety ratio for directconjugation of protein is from about 100:1 to about 500:1 moles CDAP per100 kDa of the carbohydrate-containing moiety. In another preferredembodiment, the CDAP to carbohydrate-containing moiety ratio forindirect conjugation of protein using a spacer is from 10:1 to 500:1moles CDAP per 100 kDa of carbohydrate-containing moiety. Depending onthe nature of the moieties and the conditions used, different moietyratios may be optimal.

Unreacted CDAP and reaction by-product such as dimethylaminopyridine canbe removed before derivatization or coupling to protein using a suitablepurification technique, preferably under acidic conditions, such asdialysis, ultrafiltration, or absorption to suitable bioprocessing beadssuch as SM4 beads (BioRad). Purified activated polysaccharide can alsobe prepared by precipitation, e.g., with cold ethanol.

In a preferred embodiment, a carbohydrate-containing moiety that hasbeen activated using CDAP is directly conjugated to the second moiety toproduce an immunogenic construct. In another preferred embodiment of theinvention, the carbohydrate-containing moiety which has been activatedis covalently linked to a suitable bifunctional or heterofunctionalreagent. Examples of such functional reagents include ethylene diamine,1,6-hexane diamine, adipic dihydrazide, cystamine, lysine, glutamicacid, thiol hydrazides, and thiol amines, suitably protected asnecessary. See Wong et al., "Chemistry of Protein Conjugate andCrosslinking," CRC Press (1991). The second moiety is then covalentlylinked to the functional reagent, which has already been covalentlylinked at its other terminus to the carbohydrate-containing moiety.

A preferred pH range for the coupling reaction is from about 7 to about9, more preferably about 7 to about 8.5. For conjugating apolysaccharide such as dextran, the pH is preferably from about 7.4 toabout 8.

A polysaccharide is conjugated to a protein at a ratio in the range offrom about 1:1 to about 3:1, e.g., 1:1, using CDAP in one preferredembodiment. For optimal results, high polysaccharide concentrations areavoided. Preferred constructs include tetanus conjugated to aPneumococcal polysaccharide and tetanus conjugated to Haemophilusinfluenzae PRP. Other preferred conjugates prepared according to theinvention include TT-PRP, Pn14-TT, Pn23-TT, malaria-derivedpeptide-Pn14, DT-Pn14, Pn6-TT, Pn19-TT, and peptide-TT-Pn.

In a preferred embodiment, triethylamine (TEA) is used to facilitate thecyanylation reaction, which may proceed via the formation of anintermediate Von Braun complex. TEA can be replaced by other tertiaryamines capable of forming a Von Braun complex. J. Von Braun, Chem. Ber.,33:1438 (1900).

For certain conjugation reactions, glycine, amino ethanol, or otheramino-containing reagents may be used to quench the reaction. Suchquenching reagents may also be used as one way to modify the net chargeof the conjugate.

In another embodiment, the invention relates to vaccines that are madeup of an immunogenic construct together with a pharmaceuticallyacceptable medium or delivery vehicle. Such vaccines will contain aneffective therapeutic amount of the immunogenic construct together witha suitable amount of vehicle so as to provide the form for properadministration to the patient. These vaccines may comprise alum or otheradjuvants.

Exemplary pharmaceutically acceptable media or vehicles are sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil, and the like. Saline is a preferred vehicle when thepharmaceutical composition is administered intravenously. Aqueousdextrose and glycerol solutions can also be employed as liquid vehicles,particularly for injectable solutions. Suitable pharmaceutical vehiclesare described in E. W. Martin, Remington's Pharmaceutical Sciences,specifically incorporated herein by reference.

The vaccines that may be prepared in accordance with the inventioninclude, but are not limited to, those listed in the chart below:

Chart

Diphtheria vaccine

Pertussis (subunit) vaccine

Tetanus vaccine

H. influenzae type b (polyribose phosphate)

S. pneumoniae, all serotypes

E. coli, endotoxin or J5 antigen (LPS, Lipid A, and Gentabiose)

E. coli, O polysaccharides (serotype specific)

Klebsiella, polysaccharides (serotype specific)

S. aureus, types 5 and 8 (serotype specific and common protectiveantigens)

S. epidermidis, serotype polysaccharide I, II, and III (and commonprotective antigens)

N. meningitidis, serotype specific or protein antigens

Polio vaccine

Mumps, measles, rubella vaccine

Respiratory syncytial virus

Rabies

Hepatitis A, B, C, and others

Human immunodeficiency virus I and II (GP120, GP41, GP160, p24, others)

Herpes simplex types 1 and 2

CMV (cytomegalovirus)

EBV (Epstein-Barr virus)

Varicella/Zoster

Malaria

Tuberculosis

Candida albicans, other candida

Pneumocystis carinii

Mycoplasma

Influenzae viruses A and B

Adenovirus

Group A streptococcus

Group B streptococcus, serotypes, Ia, Ib, II, and III

Pseudomonas aeroginosa (serotype specific)

Rhinovirus

Parainfluenzae, types 1, 2, and 3

Coronaviruses

Salmonella

Shigella

Rotavirus

Enteroviruses

Chlamydia trachomatis and pneumoniae (TWAR)

Cryptococcus neoformans

The invention also relates to the treatment of a patient byadministration of an immunostimulatory amount of the vaccine. The term"patient" refers to any subject for whom the treatment may bebeneficial, and includes mammals, especially humans, horses, cows, dogs,and cats, as well as other animals, such as chickens. An"immunostimulatory amount" refers to that amount of vaccine that is ableto stimulate the immune response of the patient for the prevention,amelioration, or treatment of diseases. The vaccine of the invention maybe administered by any suitable route, but is preferably administered byintravenous, intramuscular, intranasal, or subcutaneous injection.

The invention also relates to a method of preparing an immunotherapeuticagent against infections caused by bacteria, viruses, parasites, fungi,or chemicals by immunizing a patient with the vaccine described above sothat the donor produces antibodies directed against the vaccine.Antibodies may be isolated or B cells may be obtained to later fuse withmyeloma cells to make monoclonal antibodies. The making of monoclonalantibodies is generally known in the art (see Kohler et al., Nature,256:495 (1975), specifically incorporated herein by reference). As usedherein, "immunotherapeutic agent" refers to a composition of antibodiesthat are directed against specific immunogens for use in passivetreatment of patients. A plasma donor is any subject that is injectedwith a vaccine for the production of antibodies against the immunogenscontained in the vaccine.

EXAMPLE 1 Derivatization of a Carbohydrate-Containing Moiety with aSpacer

Materials:

CDAP, pyridine, hexane diamine, sodium borate, HEPES, and triethylamine(TEA) were purchased from Aldrich (Milwaukee, Wis.). Thecarbohydrate-containing moiety, T2000 dextran, with an average molecularweight of 2000 kDa, was obtained from Pharmacia (Piscataway, N.J.).

A stock of CDAP in dry acetonitrile at 100 mg/ml was stored at -20° C.and kept on ice when in use. T2000 dextran was made up at 10.5 mg/ml insaline plus 0.02% azide. Aqueous triethylamine stock was made up at 0.2Mand kept on ice during use.

Hexane diamine was made up at 0.5M in 0.1M sodium borate.

Amino group determination was made using trinitrobenzene sulfonate(TNBS) and an extinction coefficient of 11,000 m⁻¹ at 366 nm. Franci etal., J. Imm. Methods., 86:155 (1986). Carbohydrate was assayed by themethod of M. Monsigny et al., Anal. Chem., 175:525 (1988), using T2000dextran as the standard.

Control Reactions:

The following experiments demonstrate the importance of the componentsused in the derivatization reaction of the invention. The results showthat the amino groups in the final conjugate are covalently linked tothe carbohydrate and their presence is not due to artifact or"carryover" of reagent into the final product. Reactions were carriedout on ice. For trials performed, omission or substitution of reagentswas as indicated in Table 2.

In the procedure using all reagents (line 1 of Table 2), CDAP was addedto a vortexed solution of 300 μl dextran (3.1 mg) and returned to theice bucket. Thirty seconds later, the TEA was added to the vortexedsolution. Two minutes after the CDAP was added, 200 μl of the diaminewas added and the solution kept on ice for another hour. Samples weredialyzed overnight, filtered with a Millex GV filter, and furtherdesalted on a 1×15 cm P6DG column (BioRad).

As shown in Table 2 below, amino groups were optimally incorporated intodextran in the presence of dextran, CDAP, TEA, and hexane diamine. Thedata in Table 2 further demonstrate that the amino groups detected arenot due to carryover of unconjugated reagents into the final products.Although these results show that TEA is not necessary forderivatization, they show less derivatization when TEA is not present(probably due to a low pH, as later discussed).

                  TABLE 2    ______________________________________                        100        0.5 M                        mg/l  0.2 M                                   Hexane 0.1 M NH.sub.2 /    #   Saline  Dextran CDAP  TEA  Diamine                                          Borate                                                Dextran*    ______________________________________    1   0       300 μl                        15 μl                              15 μl                                   300 μl                                          0     64    2   300 μl                0       15 μl                              15 μl                                   300 μl                                          0     0    3   0       300 μl                        0     15 μl                                   300 μl                                          --    0    4   0       300 μl                        15 μl                              0    300 μl                                          --    2.1    5   0       300 μl                        15 μl                              15 μl                                   0      300 μl                                                0    6   300 μl                0       15 μl                              0    0      0     0    ______________________________________     *Moles NH.sub.2 per 100 kDa dextran.

Derivatization of T2000 Dextran with Hexane 1,6-Diamine

This experiment demonstrates that CDAP can be used to derivatizecarbohydrates to introduce amino groups at both high and low ratios.Dextran T2000 was used as a model carbohydrate. Dextran is a polymermade up of glucose monomers.

The first step in the preparation of many conjugate vaccines is theaddition of a spacer (Dick & Beurret, "Glycoconjugates of BacterialCarbohydrate Antigens," Conjugate Vaccines, J. M. Cruse & R. E. Lewis(eds.), Vol. 10, pp. 48-114 (1989)). This series of experiments,summarized in Table 3, emphasizes the ease with which a spacer can beadded to polysaccharides.

                                      TABLE 3    __________________________________________________________________________                      10.sup.-3 mole      Dextran          CDAP              TEA                 Diamine                      CDAP/mole                            NH.sub.2 /*                                % Efficiency                                        % ***    # (μl)          (μl)              (μl)                 (μl)                      Dextran                            Dextran                                (NH2/CDAP) **                                        Derivat'd    __________________________________________________________________________    1 600  5   5 600  .68   17  50.0    3.1    2 600 10  10 600  1.36  33  48.5    5.9    3 600 15  15 600  2.03  25  24.8    4.6    4 300 15  15 200  4.06  30  16.7    6.1    5 300 30  30 200  8.12  48  11.8    8.2    6 300 60  60 200  16.24 84  4.2     6.2    7 300 120 120                 200  32.48 112 6.9     20.4    8 300 15  15 200  4.06  38  18.7    6.9****    9 300 30  30 200  8.12  62  15.3    11.3****    10      300 60  60 200  16.2  35  4.3     6.4****    11      600 15  15 600  2.03  19  18.8    3.5    __________________________________________________________________________     * Moles NH.sub.2 per 100 kDa of dextran.     ** To calculate this value, NH.sub.2 /dextran values were divided by mole     CDAP/mole dextran values and multiplied by 100%.     *** Percent of glucose units within dextran bound to an NH.sub.2 group.     **** Experiment carried out at room temperature.

The experiment was conducted at two temperatures. In the runs summarizedin lines 1-7 and 11 of Table 3, all reagents were ice-cold, and in theruns summarized in lines 8-10, the reagents were at room temperature.Procedures and reagents were used as described above for the experimentsummarized in Table 2, and reagent amounts added were as indicated inTable 3. In the run represented by line 11, diamine was added in 0.15MHEPES. The reaction was slightly less efficient at lower pH. In anotherembodiment, hexane diamine was made up in 0.1M borate, pH 9.

Efficiency is defined as the number of moles of spacer groupsincorporated per mole of CDAP used, expressed as a percentage. The lastcolumn (% derivatized) is the percent of the glucose monomer units ofthe dextran which have been modified with a spacer.

The results are further illustrated in FIG. 4, which shows the totalnumber of amino groups (e.g., the spacer reagent added) incorporatedversus the moles of CDAP added per moles dextran unit. When this dataare converted into NH₂ incorporation versus moles CDAP/mole dextran, itis evident that a CDAP:glucose ratio of less than one is sufficient forhigh levels of NH₂ incorporation. Thus, minimal modification of dextranpolysaccharide is necessary for high NH₂ -group incorporation.

Furthermore, since an undetermined amount of the active cyanate ester ishydrolyzed without adding a spacer, the CDAP/glucose ratio is anoverestimate of the degree of modification of the polymer. Thus, theactual degree of modification is less than the calculated CDAP/glucoseratio.

The degree of incorporation of spacer groups at the lowest reagent dosetested (line 1), 3.1%, is comparable to that used for the synthesis ofconjugate vaccines (Chu et al., Inf. & Imm., 40:245 (1983); Dick &Beurret, "Glycoconjugates of Bacterial Carbohydrate Antigens," ConjugateVaccines, J. M. Cruse & R. E. Lewis (eds.), Vol. 10, pp. 48-114 (1989).

The table and figure demonstrate the high efficiency of the CDAPreaction for adding spacer reagents. Further optimization of reactionconditions can increase efficiency. Also illustrated is the very highlevel of incorporation of spacer groups into polysaccharide which ispossible using CDAP. At the highest amount of CDAP added (line 7),approximately 1 in 5 of the glucose units was modified (20%) with aspacer. It is not possible to obtain this degree of incorporation ofspacer with cyanogen bromide (Kagedal & Akerstrom, Acta Chemica Scan.,25:1855 (1971)).

During the reactions, there was no evident precipitation of the dextranpolysaccharide. In contrast, aggregation and precipitation of thepolysaccharide can be a problem with the cyanogen bromide method(Kagedal & Akerstrom, Acta Chemica Scan., 25:1855 (1971)).

These reactions were done in small volumes (<1 ml), thus allowing manytrial experiments to be conveniently performed. This is important whenoptimizing a procedure without wasting valuable carbohydrates andproteins. Thus, from the small volumes of reagents exemplified as wellas other information set forth herein, the artisan can readily practicethe invention using larger amounts as desired in any scale-up forcommercial use. In contrast, it is difficult to conveniently work withvery small amounts of cyanogen bromide due to its poor water solubility,uncertain potency, and toxicity.

Moreover, comparing lines 8-10 of Table 3 with lines 1-7 and 11, itappears that the level of incorporation of amino groups into dextran wasapproximately the same when the coupling reaction was carried out at 0°C. or room temperature.

Demonstration of Efficiency of Conjugation Reaction Using CDAP andVerification of Conjugation Using Radiolabeled Protein

Since the conjugation reaction using CDAP caused some absorbance at 280nm, the wavelength normally used to estimate protein concentrations,radiolabeled protein was directly conjugated to dextran. This allowedindependent determination of the protein concentration from its specificactivity. The yields and recovery of protein were determined.

BSA was lightly radiolabeled with N-hydroxysuccinimide (³H-2,3)-propionate (Amersham), essentially as described by Brunswick etal., Journal of Immunol., 140:3364 (1988). Radiolabeled BSA was dialyzedexhaustively into PBS+0.02% azide and subjected to gel filtrationchromatography on a S100HR column (Pharmacia) to remove aggregates andconcentrated by ultrafiltration using a YM30 filter (Amicon). The BSAconcentration was 21 mg/ml, determined from its extinction coefficientat 280 nm (44,000M⁻¹). The specific activity of the stock solution,determined by liquid scintillation counting, was 5.48×10¹² cpm/mole.

Other reagents were as follows: T2000 dextran (approximately 2000 kDa)(Pharmacia) was dissolved at 10.5 mg/ml in water. CDAP was made up at100 mg/ml in dry acetonitrile, triethanolamine (TEA) was made up at 0.2Min water. Glycine (pH 5.0) was prepared at 1M in water.

Protocol: Reagents were kept on ice and all reactions were performed onice. The reaction mixture was vortexed during each addition. Twenty-fiveμl of CDAP was added to 0.5 ml of dextran (5.25 mg), and 30 secondslater 25 μl TEA was added. After a total of 2.5 minutes, 5.25 mg ofradioactive BSA was added. Thirty minutes later, the reaction wasquenched by the addition of 100 μl of glycine solution and leftovernight at 4° C. An aliquot of 0.6 ml was then filtered using a Spin-Xmembrane (COSTAR). A comparison of the radioactivity aliquots before andafter filtration demonstrated that essentially 100% of the radioactivitywas recovered in the filtrate. Five hundred μl of the filtrate wasapplied to a 1×57 cm S400SF gel filtration column (Pharmacia) which wasequilibrated with saline plus 0.02% azide, and run at 0.2 ml/min.Fractions of 0.89 ml were collected and analyzed. Dextran concentrationswere determined by the method of Monsigny et al. using absorbance at 480nm. The radioactivity of a 50-μl aliquot taken from each tube wasdetermined by liquid scintillation counting, and ³ H-BSA concentrationwas calculated using its specific activity. The position of unconjugatedBSA in the column elution was determined in an independent column run.

As shown in FIG. 5, a large portion of the BSA, represented by the cpm,is in a high molecular weight form which runs in an identical positionas the dextran, represented by OD480. There is a small residual BSA peakrepresenting unconjugated protein. Table 4 contains the purificationdata.

                  TABLE 4    ______________________________________    Total protein recovered                           3.0 mg    Protein applied to column                           2.9 mg    Recovery               103%    Protein in high MW form                           >2.0 mg (68%)    (tubes 15-23)    Ratio of BSA to DEXTRAN for                           26    2000 kDa dextran    ______________________________________

The column did not cleanly separate the dextran-BSA conjugate from theunconjugated protein. This is not unusual since the high molecularweight polymers frequently cause tailing in gel filtration columns.Furthermore, since the T2000 dextran was unfractionated, it contained aspectrum of sizes. To estimate the amount of conjugated BSA in theregion where free and bound BSA overlap, a constant ratio of bound BSAto dextran was assumed. Total conjugated BSA, calculated by multiplyingthe BSA:dextran ratio×the total molar amount of dextran, was determinedas 2.55 mg. This indicates that 87% of the protein was converted toconjugate form.

                  TABLE 5    ______________________________________    Mole CDAP/              mole TEA/              % BSA    mole glucose              mole CDAP    BSA/dextran                                     Conjugated    ______________________________________    0.39      1:2          26        87    0.39      2:1          10        34    0.16      1:2          9         28    0.16      5:1          1         3    ______________________________________

The results of this BSA-dextran experiment are summarized in Table 5(line 1) along with three other trials using different amounts of CDAPand TEA (lines 2-4). Both the amount of TEA and the amount of CDAP helpget high protein to polysaccharide ratios via direct conjugation. Theoptimal reagent quantities can easily be determined since the methodpermits convenient experimentation with small amounts.

It should be emphasized that the direct conjugation reaction does notmodify the unconjugated protein, unlike the carbodiimide orheteroligation coupling methods, nor does it use harsh conditions. Thus,one could recover the unconjugated protein for further use. Since manyprotein antigens are valuable, this is a major advantage of the directconjugation method.

EXAMPLE 2A Preparation of PT-Pn14 Conjugates

The purpose of these experiments is to: (1) demonstrate that thetransformation of the protein from a low molecular weight form to a highmolecular weight form is a result of direct conjugation of the proteinto the carbohydrate; (2) determine, under one particular set ofconditions, the minimum amount of cyanylating reagent needed toconjugate the protein; and (3) demonstrate that clinically relevantconjugates can be prepared using the method of the invention.

Pertussis toxoid (PT) (from Mass. Public Health Biol. Labs, Boston,Mass.) was dissolved at 0.289 mg/ml in 0.5M NaCl, 0.02M sodiumphosphate, pH 8.8. One tenth ml of 0.1M sodium borate, pH 9.1, or 0.75MHEPES, pH 7.5, was added per milliliter of PT. Pneumococcal-type 14(Pn14) (ATTC lot 83909) was dissolved at 5 mg/ml in 0.15M saline with0.02% azide. Triethylamine (TEA) was dissolved at 0.2M in water. CDAPwas dissolved at 100 mg/ml or 10 mg/ml in acetonitrile (made up andstored at -20° C.). Glycine was made up at 1.0M, pH 5.0. Amino ethanolor other amino reagents can be substituted for glycine/HCl.

Experiment 1--Synthesis of Useful Vaccine Construct with DirectConjugation: PT-Pn14

Each tube contained 250 μg of Pn14 (50 μl) on ice. At time zero, variousamounts of CDAP as indicated in the table were added, and 30 secondslater 25 μl of TEA was added. Two minutes later 1 ml of PT was added.After about 1 hour, 100 μl of glycine solution was added.

Samples were kept at 4° C. overnight. The next day, they were filteredwith a Costar 0.45 micron spin filter and run on an HPLC TSK-gelfiltration column in 0.2M KCl. Percent HMW is the area of the highmolecular weight OD280 conjugate peak versus the OD280 peak indicatingunconjugated moiety. It is defined by (percent area void volume peak)/(%area void vol. peak+% area unconjugated moiety peak). The percent areas,obtained from the HPLC runs, were as follows:

                  TABLE 6    ______________________________________    Direct Conjugation Of Pertussis Toxoid To Pn14    #      μmole CDAP/100 kDa Pn14                                % HMW    ______________________________________    1      1720                 100.0    2      520                  52.3    3      172                  32.8    4       51                  31.0    5       17                  28.1    6      0 (PT control)       22.0    7      0; no TEA, no PT, (Pn14 control)                                --    8      0; no TEA, no Pn14; PT without Borate                                11.3    ______________________________________

Because the PT control has a HMW of 22%, there may be a small amount ofaggregation of the PT caused by the reaction conditions. This set ofdata also indicates that by varying the CDAP to polysaccharide (Ps)ratio, it is possible to control the ratio of protein to carbohydrate inthe final conjugate.

Experiment 2--Conjugation of a Monosaccharide to PT

In this series, 150 μl of a solution of 10 mg/ml glucose, which ismonomeric, was substituted for the Pn14 polysaccharide. Conditionssimilar to Experiment 1 were used except that the PT was made up inHEPES (pH 7.5, M 0.075) buffer instead of borate. Also, 20 μl instead of25 μl TEA was used. These conditions yielded the following:

    ______________________________________    #       Condition         % HMW form    ______________________________________    1       PT only, no CDAP or TEA                              <20%    2       CDAP, TEA (no glucose); + PT                              ˜0    3       Glucose, CDAP, TEA; + PT                              ˜0    ______________________________________

Numbers 2 and 3 indicate that CDAP does not polymerize the pertussistoxoid itself and that, therefore, the conversion of the PT to a highmolecular weight form is due to its coupling to the high molecularweight polysaccharide and not due to polymerization of the protein. Itwas evident from the HPLC run that glucose was conjugated to PT becausethere was a slight increase in the molecular weight of PT.

Experiment 3--Synthesis of Useful Vaccine Construct Via a Spacer:PT-Pn14

Pn14-derivatized with hexane diamine was prepared as follows. Ten μl ofCDAP (100 mg/ml in acetonitrile) was added (193 mole CDAP per 100 kDa ofpolysaccharide). Thirty seconds later 20 μl of TEA (0.2M) was added.After a total of 2.5 minutes had elapsed, 300 μl of 0.5M hexane diaminein 0.1M sodium borate (pH 9.1) was added. After one hour, the solutionwas dialyzed into water, filtered, and desalted into saline on a P6DG(BioRad) column. The void volume was pooled and concentrated with aCentricon 30 device (Amicon). It was determined to have 33 amino groupsper 100 kDa of Pn14 polysaccharide.

Pertussis toxoid was conjugated to the amino-Pn14 using heteroligationchemistry (Brunswick et al.). Fifty μl of 0.75M HEPES buffer (pH 7.5)was added to 0.44 ml of the amino-Pn14. It was iodoacetylated with 10 μlof 0.1M iodoacetyl propionate N-hydroxy-succinimide (SIAP). Pertussistoxoid was thiolated with a 20-fold molar excess of SATA (Calbiochem, LaJolla, Calif.). Each was desalted into saline, mixed, and 1/9 volume ofbuffer containing 0.75M HEPES, 10 mM EDTA, and 0.5M hydroxylamine wasadded. The final volume was 1.1 ml. After an overnight incubation, thesolution was made 0.2 mM in mercaptoethanol for one hour and then 10 mMin iodoacetamide for 10 minutes, following which it was fractionated ona S400SF gel filtration column (Pharmacia) (see FIG. 6). The void volumepeak was pooled and concentrated by pressure filtration on a PM10membrane (Amicon). Approximately 50% of the pertussis toxoid wasrecovered in conjugate form. The final conjugate contained 0.7 moles PTper 100 kDa of Pn14 polysaccharide. Protein concentration in theconjugate was determined by the Bradford assay (BioRad) using PT as thestandard. Polysaccharide concentration was determined by the method ofMonsigny et al. using Pn14 as the standard.

EXAMPLE 2B Direct Conjugation of a Protein to Pn14 Using CTEA

CTEA offers the advantage of having fewer side reactions than CDAP andleads to purer products, as described in Kohn et al., Anal. Biochem,115:375 (1981). Its disadvantage is that it is moisture sensitive, mustbe weighed out in a closed vessel, and cannot easily be prepared as astock solution.

One ml of Pneumococcal type 14 polysaccharide (Pn14) (5 mg/ml in saline)is kept at 0° C. CTEA (Available from Aldrich Chemical, Milwaukee, Wis.)is stored under dry nitrogen. Two CTEA is weighed out in a closedweighing vessel and added to the cooled, vigorously mixed Pn14. Twentyμl of TEA (0.2M in water) is immediately added while mixing. Sixtyseconds later, 5 mg of pertussis toxoid (1.5 mg/ml) is added to thestirred solution. One-half hour later, the reaction is quenched with 200μl 1M glycine (pH 5.0). After an additional hour, the solution isfiltered and passed over an S400SF gel filtration column, equilibratedwith saline. The void volume peak is collected and sterile filtered. A1:1 conjugate is produced.

Addition of Spacer Reagent to Pneumococcal Type 14 Polysaccharide UsingCTEA

One ml of Pn14 (5 mg/ml in saline) is kept at 0° C. CTEA (available fromAldrich Chemical, Milwaukee, Wis.) is stored under dry nitrogen. One mgCTEA is weighed out in a closed weighing vessel and added to the cooled,vigorously mixed Pn14. Immediately 20 μl is added to TEA (0.2M in water)while mixing. Sixty seconds later, 300 μl of 0.5M hexane diamine in 0.1Mborate (pH 9) is added while mixing. After one hour, the solution isexhaustively dialyzed into saline and sterile filtered. Since a ratio of187 mole CTEA per 100 kDa Pn14 is used, a conjugate with approximately18 amines per 100 kDa of Pn14 is produced.

EXAMPLE 3 Direct Conjugation of Pertussis Toxoid to HaemophilusInfluenzae Polysaccharide (PRP)

PRP, average MW 350 kDa, was obtained from the Massachusetts PublicHealth Biological Laboratory. Pertussis toxoid was from the same source.Fifteen μl of CDAP (100 mg/ml) was added to 100 μl (2 mg) of PRP on ice.Thirty seconds later, 30 μl of TEA was added. This represented 319 molesof CDAP per 100 kDa of PRP. After an additional two minutes, 0.75 ml ofpertussis toxoid (1.1 mg) was added. Forty minutes later, 200 μl of 1Mglycine (pH 5.0) was added to quench the reaction. After one additionalhour, the solution was passed over an S400SF gel filtration columnequilibrated with saline (see FIG. 7). The void volume was pooled andsterile filtered. The product was determined to have 1.1 PT per 100 kDaof PRP with an overall yield of 68%.

The vaccine prepared by Chu et al., Inf. & Imm., 40:245 (1983), used 377moles cyanogen bromide per 100 kDa of PRP and had ratios of 1.4 to 2.1PT per 100 kDa of PRP with yields of less than 50%. Thus, the directconjugation method of the invention yielded a similar conjugate but withless work, higher yields, and without the use of a toxic reagent.

Since many published protocols for preparing PRP conjugates start withthe PRP derivatized with a spacer (Chu et al., Schneerson et al., J.Exp. Med., 152:361 (1980); Dick & Beurret, "Glycoconjugates of BacterialCarbohydrate Antigens," Conjugate Vaccines, J. M. Cruse & R. E. Lewis(eds.), Vol. 10, pp. 48-114 (1989)), CDAP was also used to add a spacerto PRP. The conditions used were as described above but 100 μl of 0.1Mhexane diamine in 0.1M borate was added instead of the pertussis toxoid.The product was dialyzed into saline. It was determined to have 102amino groups per 100 kDa of PRP. Since this is a higher ratio than usedin published procedures, even less CDAP could have been used.

EXAMPLE 4 Immunogenic Constructs Useful as Vaccines Prepared Using CDAPChemistry Conjugation Using CDAP and a Bifunctional Reagent

In brief, a malaria-derived peptide, p28 (SEQ. ID. NO.:1: Cys Asn IleGly Lys Pro Asn Val Gln Asp Asp Gln Asn Lys), from the gamete-specificprotein pfs25, was conjugated to tetanus toxoid (TT). P28 has been shownto induce malaria transmission blocking antibodies. CDAP was then usedto couple p28-TT to Pneumococcal-type 14 (Pn14) polysaccharide.

FDA-approved tetanus toxoid was dialyzed overnight into HEPES buffer andreacted with a 30-fold molar excess of the iodoacetylating agent (SIAP).After 3 hours, reagents were removed by ultrafiltration using a Macrosep30 (Filtron Technology) and washed into fresh HEPES, 0.15M, pH 7.5,buffer. Tritium-labeled p28 was added as a solid to the derivatized TTwhile gently mixing. Following overnight reaction at 4° C., the mixturewas treated with 0.2 mM mercaptoethanol to block any remaining activegroups and then desalted on a P6DG column equilibrated with HEPESbuffer. From the specific activity of the peptide, the product wasdetermined to contain 20 moles p28 peptides/mole of TT. The conjugatewas dialyzed into saline and sterile filtered.

Direct Conjugation Using CDAP

Pn14 (obtained from American Tissue Type Collection, ATTC) has a highmolecular weight (c.a. 10⁶ daltons). P28-TT was directly conjugated toPn14 as follows. CDAP (10 μl from a 100 mg/ml stock solution inacetonitrile) was added to Pn14 (1.1 mg in 150 μl saline). Thirtyseconds later, 20 μl of triethylamine (0.2M) was added. Two minuteslater, 0.55 mg (in 0.8 ml saline) of p28-TT was added, and one hourlater, the reaction was quenched for another hour with 200 μl 1.0Mglycine (pH 5). The conjugate was then passed over an S400SF gelfiltration column equilibrated with saline and the void volumecontaining the conjugate was pooled. FIG. 9 indicates that virtually allof the p28-TT was found in the void volume in conjugated form.

Immunoreactivity of Immunogenic Constructs

Groups of 5 DBA/2 mice were immunized with i.v. with 10 μg p28-TT or(p28-TT)-Pn14 conjugate in saline, bled three weeks later, and the seraassayed by ELISA (enzyme-linked immunoabsorbent assay) for reactivityagainst recombinant pfs25 protein. Peptide p28 is derived from pfs25.Another set of mice was immunized with the same antigens precipitatedwith the adjuvant, alum (Imject, Pierce Chemical Co., Rockford, Ill.).

Consistent with the related applications, Table 7 shows that only thehigh molecular weight conjugate elicited good anti-protein titers.

                  TABLE 7    ______________________________________    Anti-pfs25 IgGl Titers    Antigen         i.v. (saline)                              s.c. (alum)    ______________________________________    (p28-TT)-Pn14   36        346    p28-TT          <10       >10    ______________________________________

This demonstrates that the CDAP method can be used to prepare usefulvaccine constructs. It also illustrates the ease with which usefulconjugates can be prepared.

EXAMPLE 5 Biologically Active Multivalent Protein Constructs PreparedUsing CDAP

To demonstrate that conjugates prepared using CDAP to directly coupleproteins to polysaccharides could yield a multivalent product (which asset forth in the related applications has enhanced immunogenicity) andthat the process could be gentle enough to preserve biological activity,various conjugates of a monoclonal antibody with dextran were prepared.These experiments used monoclonal antibody Hδ^(a) /1 with an anti-IgDantibody which crosslinks membrane IgD on B lymphocytes and inducesproliferation (Brunswick et al., Journal of Immunol., 140:3364 (1988)).As described by Brunswick et al., conjugation of multiple copies ofHδ^(a) /1 to a high molecular weight polymer such as 2000 kDa dextran(Hδ^(a) /1-AECM dextran) induced B-cell proliferation at 1000-fold lowerconcentrations and induced higher levels of proliferation thanunconjugated Hδ^(a) /1. In Brunswick et al., a simple, straightforwardbut multistep, multi-day procedure was required to prepare theconjugate. Aminoethyl carboxymethyl dextran (AECM dextran) was preparedfirst as described in Brunswick et al. and then heteroligation chemistrywas used to couple the Hδ^(a) /1 to the carbohydrate.

Hδ^(a) /1-dextran was prepared by both direct conjugation using CDAP andindirect conjugation using a spacer and CDAP as follows.

Direct conjugation: To a vortexed solution of 3.2 mg of T2000 dextran(Pharmacia) in 0.3 ml saline, 15 μl of CDAP was added (from a 100 mg/mlstock in acetonitrile). Thirty seconds later, 15 μl of 0.2M TEA wasadded while vortexing. After an additional 2 minutes, 6 mg Hδ^(a) /1 (in362 μl 0.05M sodium borate and 0.075M NaCl) was added while gentlyvortexing. After 15 minutes, the reaction mixture was quenched by theaddition of 100 μl of 1.0M glycine, pH 5.0, and passed over an S400SFgel filtration column (1×59 cm) equilibrated with saline. The columnelution is shown in FIG. 9. The void volume peak was pooled andsterilized with a Millex GV filter. The product is called Hδ^(a)/1-(CDAP)-dextran. This procedure took approximately 3 hours.

Spacer: Dextran was activated with CDAP as above (31.5 mg T2000 dextranin 3 ml saline and 25 μl CDAP followed by 25 μl TEA, 1 mole CDAP/0.06mole of glucose monomers). Three ml of 0.5M 1,6-diaminohexane in 0.1Msodium borate was added. The solution was exhaustively dialyzed intowater and then fractionated on an S400HR gel filtration column. The voidvolume was pooled and concentrated. This amino-dextran was determined tohave 147 amino groups per 2000 kDa dextran. The product is called NH₂-(CDAP)-dextran. Including dialysis, this was a two-day procedure. Incontrast, AECM-dextran usually takes about one week to prepare using theBrunswick et al. method.

Hδ^(a) /1 was conjugated to AECM-dextran and NH₂ -(CDAP)-dextran usingthe heteroligation techniques described in Brunswick et al. Theconjugates are called Hδ^(a) /1-AECM-dextran and Hδ^(a) /1-NH₂-(CDAP)-dextran, respectively. Conjugation using ACEM-dextran was atwo-day procedure.

B-cell proliferation assays, using 10,000 cells/well, were performed asdescribed by Brunswick et al. Table 8 provides the results of thoseassays, specifically indicating incorporation of tritiated thymidineinto B cells as counts per min./well.

                  TABLE 8    ______________________________________                Hδ.sup.a /1 Concentration (μg/ml)    Mitogen       1          0.1      0.01    ______________________________________    Hδ.sup.a /1-AECM-dextran                  16,045     25,774   25,850    (preparation 1)    Hδ.sup.a /1-AECM-dextran                  21,685     29,280   34,969    (preparation 2)    Hδ.sup.a /1-(CDAP)-dextran                  16,497     23,654   19,779    Hδ.sup.a /1-NH.sub.2 -(CDAP)-                  19,353     28,343   25,879    dextran    Medium (control)                    760        725      760    ______________________________________

As reported in Brunswick et al., Hδ^(a) /1 alone causes no incorporationat these concentrations. Maximum incorporation at 10-100 μg/ml Hδ^(a) /1is approximately 3000 cpm.

This data indicate that the conjugates prepared using CDAP, with andwithout a spacer, are essentially equivalent to Hδ^(a) /1-AECM dextranin their abilities to induce proliferation. Since only multivalentantibody induces high levels of proliferation at low doses, all theconjugates must be multivalent. Thus, direct conjugation with CDAP didnot affect the biological activity of the antibody. The directconjugation procedure was markedly faster to prepare than conjugatesprepared with a spacer. Further, adding the spacer and conjugating usingCDAP was much faster than preparing AECM dextran.

Thus, this experiment illustrates (1) the high yield of a multivalentconstruct using CDAP and (2) the ease and speed of preparation ofconjugates, especially direct conjugates. Conjugation using CDAP and abifunctional reagent took under 48 hours and direct conjugation tookless than three hours.

EXAMPLE 6

Unless indicated otherwise, the protocol in these experiments wasgenerally as follows. Triethylamine (TEA), acetonitrile, sulfuric acid(H₂ SO₄), resorcinol, hexane diamine, sodium borate, and HEPES wereobtained form Aldrich and were of reagent grade or better.N-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) was purchasedfrom Sigma or from Research Organics (Cleveland, Ohio). Trinitrobenzenesulfonic acid (TNBS) was obtained from Kodak Chemicals. Millex filterswere obtained from Millipore Corp.

Dextran T2000 was obtained from Pharmacia. Pneumococcal type 14polysaccharide was obtained from the ATTC (Rockville, Md.). Amino ethylcarbamyl dextran was prepared as described by Brunswick et al. MonomericBSA (bovine serum albumin) was prepared from low endotoxin Cohenfraction V BSA (Sigma catalogue #A9306) by gel filtration on a 2.6 cm×97cm S100HR column (Pharmacia), equilibrated with saline plus azide. Theproduct was shown by analytical HPLC to have less than 0.5% dimer andless than 0.1% material of higher molecular weight mass. The BSA wasperiodically checked by HPLC to confirm its monomeric status. Anextinction coefficient of 44,000M⁻¹ was used for BSA.

Polysaccharide was activated with CDAP as follows. CDAP was made up at100 mg/ml in acetonitrile and stored at -20° C. for up to one month.CDAP was slowly pipetted into a vortexed solution of polysaccharide inwater, and thirty seconds later, a volume of 0.2M TEA equal to thevolume of CDAP used was added. At 2.5 minutes, a one-fifth volume of0.5M hexane diamine in 0.1M sodium borate (pH 9.3) was added. Thereaction proceeded overnight at 4° C. The reaction product was desaltedon a P6DG or a P6 cartridge (BioRad), equilibrated with saline, and thenfurther dialyzed into saline. Some samples were concentrated using aCentricon 30 device (Amicon) and desalted again to confirm the removalof free diamine. Variations of this general procedure are indicatedbelow. The extent of derivatization with hexane diamine was determinedusing a TNBS assay for primary amines. Absorbance was measured at 366nm, using an extinction coefficient of 11,000M⁻¹ (Franci et al.).CDAP-activated dextran, derivatized using ethanolamine instead ofdiamine, was found to be TNBS negative in this assay. Polysaccharideconcentrations were determined as described by Monsigny et al. Resultsare expressed as moles of amine per 100 kDa of polysaccharide unlessindicated otherwise.

Protein conjugation to amino-dextran via a thio-ether linkage wasperformed as described by Lees et al., Vaccine, 12(3):1160, 1994.Protein was conjugated directly to polysaccharide by activating thepolymer with CDAP as described above for derivatization with amines.Protein (10 mg/ml in 0.15M HEPES, pH 7.5) was rapidly added to a gentlyvortexed solution at 21/2 minutes after the CDAP was introduced.Reactions were quenched with approximately 1/5 volume 0.5M ethanolaminein 0.75M HEPES, pH 7.5, for at least one hour before gel filtration on aS300HR or S400HR column (Pharmacia), equilibrated with saline. The peaktube from the void volume was assayed for protein with the Bradfordmethod (BioRad reagent) using BSA as the standard. Polysaccharideconcentrations were determined by the method of Monsigny et al., usingdextran as the standard. The results, which are discussed below, areexpressed as mg of protein per mg of polysaccharide unless indicatedotherwise.

Activation of Polysaccharides Using CDAP

Experiments were performed to determine whether CDAP activation ofpolysaccharides can be used to prepare conjugate vaccines underconditions that are more rapid, more gentle, more convenient, and saferthan previously reported methods. As a prototype polysaccharide, highmolecular weight dextran (T2000 dextran, Pharmacia) was activated withCDAP under a variety of experimental conditions.

From a 100 mg/ml stock solution, a volume of CDAP was slowly pipettedinto a solution of T2000 dextran in water (1.6 mg/ml as shown in FIG. 4,or 10 mg/ml as shown in FIG. 10). At 30 seconds, a volume of 0.2M TEAequal to the volume of CDAP was added, and 120 seconds later, a largeexcess of hexane diamine in sodium borate (pH 9.3) was quickly added.After desalting on a P6DG column followed by exhaustive dialysis toremove unconjugated reagents, high levels of polysaccharides were found(see FIGS. 4 and 10). Following this same procedure but in therespective absence of CDAP, the dextran, or the diamine, no amines weredetectable using the TNBS assay. Furthermore, CDAP-activated dextranreacted with a monoamine (ethanolamine), instead of the hexane diamine,was TNBS negative. To further ensure that all low molecular weightmaterial had been removed, the derivatized polysaccharide wasconcentrated by ultrafiltration and desalted a second time on a P6DGcolumn. The amine ratio was unchanged after this procedure.

The degree of derivatization was dependent on the amount ofCDAP--increases in the CDAP-to-dextran ratio led to increases in theabsolute number of amino groups substituted onto the polysaccharide asshown in FIGS. 4 and 10. The extent of derivatization was dependent onthe polysaccharide concentration for the same molar CDAP-to-dextranratio. Thus, at 1.6 mg/ml dextran, efficiencies ranged from 0.7 to 2.4percent based on moles of amines substituted per mole of CDAP, while at10 mg/ml dextran, as much as 0.2 mole of amines were substituted permole of CDAP (20% efficiency).

In order to improve the efficiency of this bimolecular reaction, thepolysaccharide concentration was increased from 1 to 50 mg/ml, using afixed amount of CDAP (see FIG. 11). At the highest polysaccharideconcentration used, more than 0.4 mole of amine was added for every moleof CDAP used. In contrast to the high level of substitution attainedwith CDAP activation, CNBr activation usually yields maximumefficiencies of about 1 to 2%.

In the absence of TEA, derivatization with diamines was markedlyreduced. To determine whether the presence of a tertiary amine such asTEA is essential for activating a soluble polysaccharide with CDAP, theefficiency of activation using TEA was compared with that usinginorganic buffer or NaOH.

One hundred μl of a CDAP solution (100 mg/ml in acetonitrile) was slowlyadded to a stirred solution of 2 ml of T2000 dextran (10 mg/ml in water)at room temperature. After thirty seconds, 1N NaOH was slowly added tomaintain the pH at about 9. After 11/2 minutes, 1 ml of BSA, 20 mg/ml in0.5M HEPES, pH 8.0, was added. After the reaction was allowed to proceedfor eighteen hours at 4° C., it was quenched by adding 100 μl of 0.5Methanolamine in 0.75M HEPES, pH 7.5. For analysis, 300 μl of the productwas gel-filtered on a 1 cm×50 cm S400HR column equilibrated with salineand azide. The void volume peak tube was assayed for protein using theBioRad assay and for polysaccharide using the resorcinol assay, and wasfound to have 0.45 mg of BSA per mg of dextran.

As shown in Table 9 below, derivatization resulted with a variety ofbuffers. Indeed, careful addition of 1N NaOH was used to raise the pH toabout nine yielded good levels of substitution.

                  TABLE 9    ______________________________________    Derivatization of dextran with hexane diamine    using various buffers    (desalted, dialyzed, concentrated, and desalted)    Buffer         NH.sub.2 /100 kDa dex    ______________________________________    TEA (0.2 M)    29    Borate (pH 8.8)                   40    Carbonate      20    NaOH           36    ______________________________________

With dextran, there were no significant differences in the levels ofderivatization over a pH range of from 8 to 10, although otherpolysaccharides have been found to be more dependent on the activationpH (see below). As noted above, if TEA is omitted and the pH is notraised, the dextran is still activated but it is derivatized to a muchlower degree. Thus, CDAP activation or coupling does not depend on thepresence of TEA or a buffer--any appropriate means may be used to raisethe pH so that the reaction mixture is sufficiently alkaline.

Table 10 shows the reaction kinetics of activation using CDAP. In theexperiment, 100 μl CDAP (100 mg/ml acetonitrile) was added to 1 mldextran (20 mg/ml) at 30 seconds, 1 ml of 0.1M sodium borate, pH 8.8,was added, and after two minutes, 0.5 ml hexane diamine in 0.75M HEPESwas added. Aliquots were desalted at the indicated times on a P6cartridge equilibrated with saline, and then exhaustively dialyzed intosaline before analysis. At high concentrations of polysaccharide andCDAP, the solutions gelled. Thus, it is more convenient to work with 10to 20 mg/ml polysaccharide solutions.

                  TABLE 10    ______________________________________    Kinetics of Reaction of CDAP-Activated    Dextran with Hexane Diamine    Reaction time NH.sub.2 /100 kDA dex    ______________________________________    15        min.    42    1         hr.     46    3         hr.     47    24        hr.     48    ______________________________________

As shown in Table 10, the derivatization reaction was rapid andessentially complete within 15 minutes. No increase in the degree ofderivatization was noted at 3 or 24 hours.

To test reproducibility, Pneumococcal polysaccharide type 14 (Pn14) wasactivated with CDAP and derivatized with hexane diamine. To a stirredsolution of 1 ml of Pn14 (10 mg/ml in water) was added 30 μl of CDAP(100 mg/ml in acetonitrile) (0.3 mg CDAP/mg Pn14). After thirty seconds,30 μl of TEA (0.2M in water) was added. At two minutes, 0.5 ml of hexanediamine (0.5M in 0.75M HEPES, pH 7.6) was added. At 11/2 hours, theproduct was desalted with P6 cartridge, concentrated by ultrafiltration,and again desalted, and then assayed for amines with TNBS and for Pn14with resorcinol/sulfuric acid. As shown in Table 11, efficiencies of13-15%, based on moles of amines detected per mole of CDAP used, wereobtained in three experiments performed over a one-year period.

                  TABLE 11    ______________________________________                            Efficiency    Experiment NH.sub.2 /100 kDA dex                            (mole NH.sub.2 /mole CDAP)    ______________________________________    A          17.9         14.1%    B          19.8         15.5%    C          17.3         13.6%    ______________________________________

The results tabulated above indicate stability of the CDAP reagent inthe freezer, reproducibility, and high efficiency. In comparison, CNBrsolution is not stable, and the CNBr-activation procedure is difficultto reproduce and has an efficiency of about 2%.

Direct Conjugation of Protein to CDAP-Activated Ps

As with derivatization of amines, the extent of protein conjugation tothe polysaccharide was dependent on the amount of CDAP used to activatethe polysaccharide. As shown in FIG. 12, at a concentration of 10 mg/mldextran, the CDAP:dextran ratio linearly increased with the BSA:dextranratio of the product. Similar ratios of BSA:dextran could also beobserved at even lower CDAP:dextran ratios if the protein and/orpolysaccharide concentrations were increased.

Control reactions performed in the absence of dextran and analyzed bygel filtration indicated that the CDAP by itself did not aggregate orpolymerize the BSA (protein). A CDAP-treated sample (0.5 ml water+25 μlCDAP @ 100 mg/ml in acetonitrile+50 μl 0.2M TEA+0.5 ml BSA @ 10 mg/ml in0.5M HEPES, pH 8.0) and a control sample (0.575 ml water+0.5 ml BSA(monomeric) @ 10 mg/ml in 0.5M HEPES, pH 8.0) were prepared. The sampleswere allowed to react overnight and were quenched with 100 μl of 0.5Methanolamine in HEPES. After quenching for one hour, the samples wererun on a S400 1 cm×50 cm column in saline and azide at 0.75 ml/minute.The OD280 over the column was summed and divided into the sum of theOD280 over the tubes preceding the BSA peak. The CDAP-treated sampleshowed 0.6% polymeric BSA, and the control sample showed 0.7% polymericBSA. Thus, the high molecular weight protein is not due toself-polymerization or aggregation.

Moreover, under normal conditions, CDAP does not crosslink thepolysaccharide. This was confirmed by the following HPLC experimentwhere 70 kDa of dextran was activated and then reacted with ethanolamineand run on a gel filtration column. Specifically, 2.5 mg T70 dextran (10mg/ml) was combined with 20 μl of CDAP (100 mg/ml). At thirty seconds,20 or 60 μl of 0.2M TEA was added, and at two minutes 100 μl of 0.5Methanolamine in 0.75M HEPES, pH 7.6, was added. After one hour, sampleswere run on a G4000 PWXL (Tosohaas) or an SEC3000 (Beckman) in 0.2M NaCland detected by refractive index (void volume for each column was about5 minutes, eluting salt at about 10 minutes). No evidence of a shift tohigher molecular weight was observed.

As the following comparative experiment shows, extreme conditions shouldbe avoided to prevent the CDAP from crosslinking the polysaccharide. Oneml of T2000 dextran (100 mg/ml water) was combined with 176 μl of CDAP(100 mg/ml). After thirty seconds, 176 μl of 0.2M TEA was added, whichyielded a gel in less than two minutes.

To determine the optimum activation time and to examine the stability ofthe CDAP-activated polysaccharide, protein (BSA) was added 5-300 secondsafter the addition of the CDAP and TEA, and the BSA:dextran ratio of theproduct was determined. The results shown in FIG. 13 suggest that theoptimal activation time is about 2 minutes and that the activatedpolysaccharide is stable over this time period. If the protein is addedat one hour, the reaction yield declines by about one third.

Aqueous mixtures of CDAP and polysaccharides were found to be stable, asreflected in FIG. 14. Sixty μl of CDAP (100 mg/ml) was added to 1 mlwater, and 100-μl aliquots of this CDAP solution were combined with 100μl of polysaccharide (dextran, 20 mg/ml) at various times over a periodof 10-300 seconds as shown in FIG. 14, followed 30 seconds later bycombination with 15 μl of a TEA solution (0.2M). Two minutes after beingcombined with the TEA, 100 μl of BSA (30 mg/ml) was added. The reactionwas quenched at 48 hours.

No significant differences were found in the finalprotein-to-polysaccharide ratios over the entire range of additiontimes. The results shown in FIG. 14 are consistent with the stability ofCDAP in acidic solutions and the observation that solutions of CDAP inwater become acidic. Thus, water can be substituted for the organicsolvent if the reagent solution is to be used the same day.Alternatively, CDAP can be added as a solid to the solution ofpolysaccharide. In working with small amounts of CDAP, it has been foundmore convenient to work with solutions than to work with the solidreagent. Furthermore, whereas the rapid addition of an acetonitrilesolution of CDAP will sometimes precipitate the polysaccharide,precipitation can be avoided if an aqueous solution of CDAP is used.Aqueous stock solutions of CDAP can be prepared at concentrations up to75 mg/ml.

FIG. 15 shows that protein conjugation to the polysaccharide wasrelatively rapid, and within three hours 80% of the maximum conjugationhad been attained. Even more rapid coupling could be achieved byincreasing the protein concentration, the polysaccharide concentration,and/or the CDAP concentration.

As indicated in FIG. 16, the pH of the reaction solution during thepolysaccharide activation is another important parameter inpolysaccharide activation with CDAP. As the pH during the activationstep was increased from 7.0 to 8.3, there was an increase inpolysaccharide activation as reflected by a marked increase in couplingefficiency. The BSA:dextran ratio of the conjugate increased 4-fold asthe pH increased from 7.0 to 8.3. At a pH higher than 8.3, there waslittle or no increase in the ratio. The pH dependence of CDAP activationexplains the low level of derivatization that was previously observed inthe absence of TEA, since the pH of a CDAP solution in water isinitially near neutral and becomes more acidic.

As was noted earlier with respect to the derivatization ofpolysaccharides with amines, a tertiary amine buffer is not necessaryduring activation of the polysaccharide for the direct conjugation ofproteins. Thus, direct conjugation of protein to polysaccharides may bedone, e.g., using a pH stat or automatic titrator to raise the pH duringthe activation step. This could be advantageous in preparing vaccineconjugates.

FIG. 17 illustrates that the pH of the reaction solution during thecoupling of the protein to the activated polysaccharide is an importantparameter in the direct conjugation of protein with CDAP. In theexperiment for which results are reported in FIG. 17, several bufferswere tested over a wide range of pH values and at a lowprotein-to-polysaccharide ratio. The protocol was as follows.

To four ml of T2000 dextran (10 mg/ml in water) was added 133 μl of aCDAP solution (100 mg/ml in acetonitrile, freshly prepared) (0.33 mgCDAP/mg dex). After 30 seconds, 266 μl of TEA (from a 0.2M stock) wasadded, and the pH reached a maximum of 9.6. After 21/2 minutes, the pHwas adjusted to 5.0 using 60 μl of 1M NaAc (sodium acetate). Fourhundred μl of activated dextran was transferred to tubes containing 200μl of BSA (15 mg/ml) (0.8 mg BSA/mg dex) and 100 μl of a buffer (1MNaAc, pH 4.7, 5.7; 0.5M HEPES, pH 6.94, 7.43, 8.15; 0.1M NaPO₄, pH 8.0,8.67; 50 mM sodium borate, pH 9.0, 9.6) (not controlled for ionicstrength). One hour after transfer, 350 μl of the solution of a tubewere combined with 100 μl of freshly prepared 0.5M ethanolamine in 0.75MHEPES (pH 7.5). Twenty hours later, 100 μl of ethanolamine were added tothe remaining solution. The reaction was quenched for at least two hoursand the product run on S300HR or S400HR columns equilibrated with salineplus azide. The peak void volume tube was assayed for BSA using theBioRad assay and for polysaccharide using the resorcinol assay.

As shown in FIG. 17, most of the protein was coupled to thepolysaccharide at a pH as low as 7.4, a substantial amount was coupledat a pH as low as 6.9, and a small but significant amount was coupledeven at a pH as low as 5.7. For the conditions of this experiment, a pHof about 8 appeared to be optimal. Although the results show that the pHof the coupling step is important, they show that coupling can be doneover a wide pH range. Since the coupling reaction is so inefficient at apH of 5, however, quenching should be done at about a pH of 7 to 8.

Increased amounts of coupling can be obtained even at low pH byincreasing the protein-to-polysaccharide ratio, the polysaccharideconcentration, and/or the amount of CDAP used. For example, by usingmore reagent or more protein, higher yields can be obtained even at a pHof 7. Thus, direct protein coupling can be achieved at a near-neutral pHusing CDAP to activate the polysaccharide.

FIG. 17 indicates that phosphate is also inhibitory to the couplingreaction, which may be due to ionic interactions or to the slightnucleophilic character of the phosphate. Increasing the amount of CDAPand the pH during the coupling, however, will increase the conjugationratio/yield. If phosphate is present during the CDAP activation,addition of the diamine is inhibited.

Phosphates of PRP and Pn6 may cause inhibition, as shown by thefollowing experiment. Twenty μl of CDAP (100 mg/ml in acetonitrile) wasadded to a vortexed solution of 2 mg Pn6 (Pneumococcal type 6, apolyribitol phosphate polysaccharide) (10 mg/ml in water). Thirtyseconds later, buffer (100 μl of 0.1M sodium borate or 40 μl of 0.2MTEA) was added. At two minutes, 100 μl of BSA (20 mg/ml) in 0.5M HEPES,pH 8, was added. After incubating overnight at 4° C., the reaction wasquenched with 100 μl of 0.5M ethanolamine in 0.75M HEPES, pH 7.5,followed by gel filtration on an S400HR column (Pharmacia) equilibratedwith saline and 0.02% azide. The peak void volume tube was assayed forprotein and polysaccharide. For comparative purposes, in trial 4 dextranwas derivatized in the same manner. The results are reported in Table 12below.

                  TABLE 12    ______________________________________    Trial  Ps         Buffer     BSA/Ps (mg/mg)    ______________________________________    1      Pn6        0.2 M TEA  0.06    2      Pn6        0.1 M sodium                                 0.16                      borate (pH 8.8)    3      Pn6        0.1 M sodium                                 0.31                      borate (pH 10)    4      dex        0.1 M sodium                                 0.77                      borate (pH 8.8)    ______________________________________

For Pn6 with the TEA buffer (trial 1), the yield was very low. As the pHwas increased with sodium borate (trials 2 and 3), the yield increased.The same conditions give much higher yields for dextran (see, e.g.,trial 4). Thus, phosphate-based polysaccharides such as Pn6 requireadjustment in the pH and/or CDAP ratio to prepare conjugates in goodyields.

The next experiment shows that the isourea bond formed by CDAPactivation is stable and robust. In this experiment, ε-TNP-lysine wascoupled to dextran via CDAP. Samples 1-5 were made up as follows:

400 μl TNP/CDAP/dex+100 μl saline (control)

400 μl TNP/CDAP/dex+100 μl 2M NaCl

400 μl TNP/CDAP/dex+100 μl 9M GuHCl

400 μl TNP/CDAP/dex+100 μl saline (reacted in incubator @ 37° C.)

400 μl TNP/CDAP/dex+100 μl saline (control)

The samples were allowed to react overnight in the dark, except example4, which was reacted as indicated. The samples were then desalted on aP6 cartridge in 10 mM sodium borate at 1.0 ml/minute. The fractions wereread at OD366 and the peak tube of the void fractions was assayed. Theresults are provided in Table 13 below.

                  TABLE 13    ______________________________________    Sample  TNP (μM) Dex (μM)                                 TNP/100 kDa dex    ______________________________________    1        96         9.7      10    2       134         12       11    3       127         11       12    4       137         13       11    5       107         10       11    ______________________________________

For each sample the TNP:dextran ratio was unchanged, indicating that theisourea bond was stable to the test conditions.

Biological Activity of Conjugates

To determine whether CDAP activation of the polysaccharide had anydetrimental effect on its ability to induce antibody responses, itsbiological activity in vitro was tested. BSA was either directlyconjugated to CDAP-activated Pneumococcal polysaccharide type 14 orcoupled to Pneumococcal polysaccharide type 14 derivatized with hexanediamine followed by iodoacetylation and reaction with thiolated protein(Lees et al.). Each conjugate had a ratio of mg BSA/mg Pn14. InbredDBA/2 mice were immunized subcutaneously with 50 μg of BSA, either freeor as a polysaccharide conjugate, in the absence of adjuvants. Sera werecollected 14 and 28 days later, and anti-BSA and anti-Pn14 antibodytiters determined by ELISA.

Neither unconjugated BSA nor unconjugated Pn14 stimulated a detectableprimary response. In contrast, the BSA-Pn14 conjugates stimulatedsignificant antibody responses to both the protein and polysaccharidecomponents, regardless of whether the protein was coupled by indirectconjugation using a spacer or by direct conjugation. Mice immunized withBSA-dextran prepared using a spacer or direct coupling to CDAP-activateddextran gave titers comparable to those obtained when conjugates wereprepared using other chemical methods. Moreover, TT-PRP conjugatesprepared using CDAP activation have shown in rats immunized with theconjugates anti-PRP responses comparable to those shown in ratsimmunized with TT-PRP conjugates prepared using CNBr activation.Furthermore, tetanus conjugated directly to CDAP-activated Pn14 had highanti-tetanus and anti-Pn14 antibody responses; opsonic assays indicatedthat these antibodies were protective.

Summary

The method of the invention utilizing CDAP represents a reproducibleapproach that can be used to activate various clinically relevantpolysaccharides, some of which are sensitive to a high pH. Activation israpid, so the time is spent at a high pH is minimized. The methodproduces highly immunogenic protein-polysaccharide conjugates, which canstimulate in mice humoral antibody to both the protein andpolysaccharide components even in the absence of adjuvant.

The variables which have been found to profoundly influence the extentof polysaccharide activation are the concentrations of CDAP andpolysaccharide, and the pH. A preferred pH for conjugating is about 7 toabout 9, more preferably about 7.4 to about 8.0, which is a range atwhich most polysaccharides are stable. Other pH ranges, e.g., a range offrom about 7 to about 10, may be more suitable for otherpolysaccharides.

By manipulating the polysaccharide and/or CDAP concentration, theefficiency of derivatization can be increased to 50%, as compared to the1-2% found with CNBr. Furthermore, a product with greater than 50 NH₂groups per 100 kDa of polysaccharide can be achieved under the preferredconditions. The method of the invention does not depend on the presenceof tertiary amines, as has been described by previous investigatorsexperimenting with CDAP. The activation of the polysaccharide is rapid.Similarly, protein conjugation to activated polysaccharide is rapid.

The invention offers the advantages of reproducibility, rapidreactivity, and perhaps most notably, the ability to easily manipulateprotein:polysaccharide ratios. For example, conjugates with variousprotein-to-polysaccharide ratios can be achieved by altering theconcentration of CDAP and/or the polysaccharide concentration and/or theprotein concentration. This may provide an approach to studying not onlythe role of protein:polysaccharide ratio in influencing the magnitude ofthe antibody response to the conjugate, but also the role of thethree-dimensional structure at a given protein:polysaccharide ratio.

The immunogenicity of the protein-polysaccharide conjugates preparedusing CDAP is significantly greater than the response demonstrated byeither of the unconjugated components. Furthermore, the antibody that isproduced is reactive with the unconjugated protein, and the response canbe boosted using the unconjugated protein as well as the conjugatedprotein. This suggests that any chemical alteration of the proteinduring conjugation has no detrimental effect on its ability to stimulateantibodies with reactivity to the native protein, nor on its ability tostimulate B cells with reactivity to the unconjugated protein.

Additionally, CDAP-activated polysaccharides can be used in preparationfor conjugation of anti-Ig antibodies. Anti-Ig-dextran conjugates induceabout 100- to 1000-fold greater activation of B cells as compared tounconjugated Ig. Anti-Ig-dextran conjugates prepared using directconjugation to CDAP-activated dextran are as effective B-cellstimulatory reagents as the conjugates prepared using otherheteroligation coupling to AECM dextran.

CDAP is useful for preparing a variety of immunological reagents, suchas biotinylated polysaccharides for ELISA and ELISA spot antigens andTNP-polysaccharides (e.g., TNP-dex, TNP Ficoll) for model Ti-2 antigens.

Thus, the inventive method, which employs CDAP to produce immunogenicconstructs such as polysaccharide-based conjugates, offers manyadvantages to the currently available technology for preparingimmunogenic constructs. It will be apparent to those skilled in the artthat various modifications in the methods and embodiments of the presentinvention can be made without departing from the scope or spirit of theinvention. Thus, the invention should not be construed to be limited bythe description and drawings, but by the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 1    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    CysAsnIleGlyLysProAsnValGlnAspAspGlnAsnLys    1510    __________________________________________________________________________

What is claimed is:
 1. In a method for preparing a vaccine comprising an immunogenic construct and a pharmaceutically acceptable medium or delivery vehicle, the improvement comprising producing the immunogenic construct by a process comprising the steps of:(a) activating a water-soluble viral or bacterial polysaccharide with an organic cyanylating reagent selected from the group consisting of 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate and N-cyanotriethyl-ammonium tetrafluoroborate to form an activated carbohydrate, said activating being carried out at a pH of from about 8 to about 10 in the presence of a buffer and at a ratio of from about 100:1 to about 500:1 moles of the organic cyanylating reagent per 100 kDa of the polysaccharide; and (b) directly coupling said activated carbohydrate to a water-soluble protein to form the immunogenic construct capable of stimulating an immune response, said direct coupling being carried out at a pH of from about 7 to about
 9. 2. A method according to claim 1, wherein said organic cyanylating reagent is 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate.
 3. A method according to claim 1, wherein said buffer is triethyl amine.
 4. A method according to claim 1, wherein the polysaccharide is conjugated to the protein at a ratio of from about 1:1 to about 3:1.
 5. A method according to claim 1, wherein the concentration of the polysaccharide is from about 1 to about 20 mg/ml.
 6. A method according to claim 1, wherein the polysaccharide is selected from the group consisting of Pneumococcal polysaccharide, Haemophilus influenzae polysaccharide, Group A streptococcus polysaccharide, Group B streptococcus polysaccharide, and N. meningitidis polysaccharide.
 7. In a method for preparing a vaccine comprising an immunogenic construct and a pharmaceutically acceptable carrier, the improvement comprising producing the immunogenic construct by a process comprising the steps of:(a) activating a water-soluble viral or bacterial polysaccharide with an organic cyanylating reagent selected from the group consisting of 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate and N-cyanotriethyl-ammonium tetrafluoroborate to form an activated carbohydrate, said activating being carried out at a pH of from about 8 to about 10 in the presence of a buffer and at a ratio of from about 10:1 to about 500:1 moles of the organic cyanylating reagent per 100 kDa of the polysaccharide; and (b) indirectly coupling said activated carbohydrate to a water-soluble protein to form the immunogenic construct capable of stimulating an immune response, said coupling being carried out at a pH of from about 7 to about 9 by steps comprising covalently joining the polysaccharide to a bifunctional or heterofunctional spacer reagent, and covalently joining the protein to the spacer reagent.
 8. A method according to claim 7, wherein said spacer reagent is selected from the group consisting of ethylene diamine, 1,6-hexane diamine, adipic dihydrazide, cystamine, glycine, and lysine.
 9. A method according to claim 7, wherein said organic cyanylating reagent is 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate.
 10. A method according to claim 7, wherein said buffer is triethyl amine.
 11. A method according to claim 7, wherein the polysaccharide is conjugated to the protein at a ratio of from about 1:1 to about 3:1.
 12. A method according to claim 7, wherein the concentration of the polysaccharide is from about 1 to about 20 mg/ml.
 13. A method according to claim 7, wherein the polysaccharide is selected from the group consisting of Pneumococcal polysaccharide, Haemophilus influenzae polysaccharide, Group A streptococcus polysaccharide, Group B streptococcus polysaccharide, and N. meningitidis polysaccharide. 